Inhibitory signaling from LFA-1 to α₄β₁ in Jβ2.7/LFA-1 cells or T blasts requires Tiam1 and an intact cytoskeleton. (A) Jβ2.7 or Jβ2.7/LFA-1 cells or (B) T blasts were treated with SDF1-α, the LFA-1 activating (CBR LFA-1/2), blocking (7E4), or neutral (CBR LFA-1/7) antibodies with or without a Tiam1 inhibitor. (C) Jβ2.7/LFA-1 cells transfected with Tiam1 siRNA or control siRNA and treated with activating (CBR LFA-1/2) or blocking (7E4) antibodies. (D) Jβ2.7/LFA-1 cells were treated with cytochalasin D (0.1 or 10 μg/mL) and LFA-1 activating (MEM48), blocking (7E4), or neutral (CBR LFA-1/7) antibodies. Adhesion to VCAM-1 under flow was quantified from 6 screens in triplicate. Amounts of bound cells and standard deviations are shown. Phalloidin staining of F-actin of Jβ2.7/LFA cells treated with 0.1 or 10 μg/mL cytochalasin D. Scale bar represents 10 μM. (E) Jβ2.7 or Jβ2.7/LFA-1 cells treated with LFA-1 activating antibody (CBR LFA-1/2) or blocking antibody (7E4) were lysed and Rac-GTP pulled down from lysates. Immunoblot shows Rac-GTP from pulldown and total Rac or actin from cell lysates. (F) Jβ2.7 or Jβ2.7/LFA-1 cells were incubated with the Rac inhibitor or left untreated and with the LFA-1 activating antibody (CBR LFA-1/2), and cells adhering to VCAM-1 under flow quantified as earlier. *P < .05; **P < .01.